STATE L,.. F,-ARD
May 1942 ET-192
United States Department of Agriculture
Bureau of Entomology and Plant Quarantine
METHOD FOR THE PREPARATION OF SPORE-DUST MIXTURES OF
TYPE A MILKY DISEASE OF JAPANESE BEETLE LARVAE
FOR FIELD INOCULATION
By S. R. Dutky,
Division of Fruit Insect Investigations
Studies at the Japanese Beetle Laboratory of the Bureau of
Entomology and Plant Quarantine, at Moorestown, N. J., have indi-
cated that the type A milky disease of Japanese beetle larvae
(Popillia japonica Newm.) may be utilized for reduction of the
larval population of this insect under field conditions. This
disease is caused by the spore-forming I:acterium Bacillus popilliae
Dutky. The procedure described herein outlines the method which
has been developed and is nowv, in use for producing the spores in
large quantities and preparing them in a suitable form for storage
and field distribution. 1/ This summary outline has already been
furnished to certain State agencies to aid them in disease-distri-
Lution projects which they were undertaking. It is now being
included in the ET series to mnke it more generally available to
those desiring information on the procedure followed in preparing
the spores of the milky disease for distribution.
(1) STOCK CULTURES OF THE ORGANISMS
It has been found that filrnis of dried blood from diseased
larvae on glass microscope slides are satisfactory as stock cul-
tures of these spores. Tests have shown these films to be suitable
for use after storage for periods as long qs 4 years. In a number
of instances pure stock cultures of known identity and tested
virulence have been furnished by the Bureau of Entomology and Plant
Quarantine to official agencies interested in work with the milky
disease of the Japanese beetle.
I/ Patents pending.
(2) PREPARATION OF THE INOCULUM
The first step is the preparation of a spore suspension to
be used as the inoculum. This is done by removing the spores from
the stock slides by moistening the dried blood films with sterile
distilled water with the aid of a sterile pipette. This is facili-
tated by stroking the moistened film with the side of the pipette
so as to bring the spore material into suspension. Then by holding
the tip of the pipette against one corner of the slide and tilting
the slide toward the pipette tip, the suspension is run into the
inclined pipette. The suspension in the pipette is then run into a
sterile test tube, and fresh distilled water is flooded over the
slide. By the same procedure the second portion of water is re-
moved from the slide and run into the test tube. Spore counts
on the suspension in the test tube are made with a counting chamber *
and the number of spores is adjusted to approximately 300 million
per cc. The details of a procedure for making this adjustment
easily and rapidly are given in the appendix (p. 6 ) following the
main body of this paper.
About one-half cc. of this adjusted suspension is drawn into
the pipevte, and the pipette is placed in a horizontal position on a
wire basket. The needle of the hypodermic syringe 2/ is inserted
into the tip of the pipette, and the suspension is drawn into the
syringe by pulling back the plunger. The syringe is then held
vertically with the needle up, and the plunger is further withdrawn,
admitting air into the syringe. While still in a vertical position
the plunger is pushed forward, driving the entrapped air upward and
out of the needle. The needle is then reintroduced into the tip
of the pipette, and the suspension is discharged into the pipette
until the plunger rests at the 0.35=cc. mark on the syringe. Ex-
treme caution must be exercised to insure that no bubbles of air
remain in the syringe, since even the smallest bubble will result
in a failure of these small volumes to be injected into the grub
because of the back pressure and compressibility of the air locked
in the syringe. The syringe is then slipped into place in the in-
jection block and the spring clip is fastened. The microinjector
with the loaded syringe in place is shown in figure 1. The micro-
meter screw is turned forward until a droplet is forced from the
end of the needle. This droplet is removed with a piece of absor-
bent cotton. An inoculating dosage is discharged into a dry,
sterile test tube by depressing the pressure bar of the ratchet
2/ The syringe is part of the special "microinjector" de-
veloped at the Japanese Beetle Laboratory by Dutky and Fest for in-
jecting larvae, and may be readily removed from the injector for
mechanism. A 1-co. portion of sterile distilled water is then
pipetted into the test tube, washing the droplet of spore suspen-
sion from the side of the test tube and suspending the spores in
this volume. Counts are made on this sample to check on the spore
(3) INJECTION OF LARVAE
In this work it is preferable to use full-grown larvae, al-
though small third-instar and even second-instar larvae may be
used if larger larvae are not available.
Larvae are injected in the following manner: The grub is
held firmly but lightly between thumb and forefinger, the dorsal
posterior portion outward, and guided toward the needle point. The
grub is forced onto the needle point so ihat the needle enters in
the dorsal portion of the suture between the second and third pos-
terior abdominal segments. Care must be taken that the needle
enters horizontally so as not to puncture the intestine. Care must
also be taken to insure that as far as possible the site of puncture
be free from adhering soil particles. The larva is then allowed
to hang suspended on the needle during the injection. The injection
is made at this time by depressing the pressure bar of the ratchet
mechanism of the microinjector, which forces an inoculating dosage
into the body cavity of the grub. This operation is illustrated in
figure 2. By volume, the dosage approximates one three-hundredth of
a cc. Since the inoculum is adjusted to 300 million spores per cc.
the resulting spore dosage approximates 1 million spores.
(4) INCUBATION OF INJECTED LARVAE
Incubating boxes having a capacity of 500 larvae are used to
hold the injected larvae during the period of incubation. These are
equipped with metal cross-section separators which divide each of 5
layers into 100 compartments. After injection, the inoculated lar-
vae are dropped into the separate compartments. As soon as all
compartments in a layer are occupied, additional soil is added to
fill completely the compartments. A flat metal separator is placed
on top of the filled layer, and a new cross section is then put in
place in the box on top of the completed layer. The compartments in
the new layer are partially filled with soil, injected larvae are
placed in these compartments, and the whole process is repeated until
all 5 layers in the box have been filled. The soil used to fill
the boxes should contain, for each 100 pounds of soil, 1/2 pound
of grass seed, which when sprouted will serve as food for the larvae
during the incubation period.
The boxes are incubated at 86 F. for from 10 to 12 days. A
high humidity should be maintained in the incubation chamber to pre-
vent excessive drying out of the soil during incubation. The incu-
bation boxes stacked in the incubating chamber are shown in figure 3.
(5) TREATMENT OF DISEASED LARVAE PRIOR TO MIXING WITH CARRIER
after incubation, the boxes are broken down, and the diseased
grubs are screened out of the soil and dropped into a battery jar of
ice water. T_., ice water inactivates the larvae, permitting thousands
of them to be placed in the jar without danger of losses of spores
due to nipping. The larvae are then washed in a colander to re-
m:0've .ihering soil particles, returned to the glass jar, packed with
ice and held in a refrigerator at a temperature of approximately
32 to 35 F. until used, as shown in figure 4.
",.en sufficient ,,'mbers of diseased larvae have accumulated,
the excess water is drained off the grubs, and the grubs are crushed
by running them through a meat chopper. After all the larvae have
been run through the chopper, the chopper is washed out with a small
quantity of water to remove the adhering grub material. The re-'
suiting suspension, together with the wash water run through the
chopper, is V.aced in a glass graduate and made up to even volume.
Counts are .i.a-.e on this suspension, using a suitable dilution
(1:2,000), and the density in spores per cc. is recorded.
(6) INCORPORATION OF THE SPORES WITH CARRIER
The standardized grub su'.-; i-.,jnsion is then added to the carrier
(calcium carbonate, precipitated, U.S.P.) so that the mixture will
contain a billion spores per gram of the dry material. The moist
dust is then mixed thoroughly by running, it through a mixing device,
such as a blade cutter or trowel mixer. After thorough mixing has
been ac o ilished, the moist dust is passed throu-i; a hi;!gh-speed
impeller-type blower, which shears the agglomerated particles.
Dry-inz. of the dust is accomplished by drawing heated air
thr,:..:p.h the blower and exposing the finely divided particles to the
warm air blast. This material when dry is the stable concentrated
spore-dust preparation. The concentrate is then mixed with suit-
able quantities of dry carrier (talcum powder, marble flour, etc.)
and stored until used. In the colonization work carried on by the
Bureau, this final mixture contains 100 million spores per gram.
Figure 5 is a photograph of the experimental plant-scale equipment
used in preparing spore-dust material.
(7) FINAL CHECK ON THE SPORE CONTENT OF THE CONCENTRATED SPO:E-DUST
The spore content of the dry spore-dust concentrate may be
checked as follows: Ten grams of the powder are placed in a volu-
metric flask, 200-ml. capacity, approximately 100 ml. of distilled
water added, the flask is shaken to wet the particles, and then
20 cc. of concentrated hydrochloric acid is added to the suspen-
sion. The flask is gently rotated with the stopper out until gas
evolution ceases. The suspension is then made up to volume (200
ml.). The flask is stoppered and sbl,.. vigorously. A loopful
of this suspension is then used to fill the counting chaiom-:r. The count
on the suspension should be 50 million spores per cc. The :-,-.;tual count
in millions obtained is multiplied by 0.02 and the result recorded
as the spore content in billions per gram of the concentrated dust
SUGGESTIONS AND PRECAUTIONS TO BE OBSERVED
1. The necessity of insuring the absence of ny air entrapped
either in the needle or syringe cannot be over&-'.-.phe3ized. Air is
easily detected by the operator and is evidenced by the formation
of a droplet on the end of the needle when the larva is withdrawn.
2. In case of accidental puncture of the intestine of a .7,rub
during injection, the hypodermic needle should be sterilized immediate-
ly by swabbing with 0.5-percent sodium hypochlorite solution irefore pro-
ceeding with further inoculation. Otherwise subsequent larvae ri.lj be
infected with septicemia-producing bacteria, contaminating the needle
from the punctured gut.
3. Syringes should be flushed imneictely after use with dis-
tilled water and then with alcohol to avoid a pl-..,ed ,,.,le or
"freezing" of the plunger in the syringe.
4. Syringes should be returned to test tubes and the plugs
replaced after use. Sterilization of glassware should be effected
as soon as possible after cleaning.
5. Careful records should be made of nu.iers of larvae in-
jected, spore and volume dosages, the nuirbLer of larvae living at the
end of the incubation period, the number diseased, the number not
infected, and the yields in number of spores per grub. F'r.:r such
records it is easy to control the process fully and corre-t any
serious faults in procedure as they occur. The saving this control
work can effect will more than offset the additional tji,., and effort
Method for preparing and adjusting density of
spore suspensions for use in the inoculation procedure
In section 2, specific directions are given for the prepara-
tion of the inocula from stock culture slides. Reference is made on
page 2 to the matter of deterLminin, the density of the spore suspen-
sion and the adjustment of the spore density to 300 million spores
per cubic centimeter. It is believed that the procedure followed
at the Japanese Beetle Laboratory in connection with these points may
be of interest to other workers and, therefore, a detailed procedure
is given herewith.
To prepare sufficient spore suspension to inoculate from 500
to 1,000 larvae, the following procedure will be found helpful. First
(A) Sample tube containing 1 cc. of sterile distilled water.
(B) Inoculum tube containing 2 cc. of sterile distilled water.
1. Wash spores from two culture slides into the inoculum
tube (B) with an additional 1-cc. portion of sterile distilled water
so that the total volume of the suspension is 3 cc.
2. Fill the capillary pipette with a loopful of this suspen-
sion to the 0.01-cc. mark. Discharge the capillary pipette into
the sample tube (A). Shake the sample tube.
3. Withdraw a loopful of the suspension in the sample tube
and fill the counting chamber. Place the counting chamber in the
mechanical stage of the microscope and allow the charn>.er to stand 2
minutes to permit settling of the spores in the chamber.
4. Using a 4-mm. N. A. 0.65 objective, count the number of
spores in 5 large squares of the Levy counting chamber (80 small
5. The nIUmber of spores in 5 large squares multiplied by 5
equals the number of spores in millions per cc. of the suspension in
6. This figure divided by 100 (pointing off two places)
equals the volume to be made up to in order to have the desired con-
centration of 300 million spores per cubic centimeter of suspension.
Subtract 3.00 to determine the volume of sterile distilled water
which must be added.
For example: A spore suspension shows a count of 86 spores
per 5 large squares. Multiplied by 5 this gives us the count as
430 million spores per cc. Or the suspension must be made up to
4.30 cc. to have the standard concentration of 300 million spores
per cc. Substracting 3, this leaves 1.30 cc., the volume of sterile
distilled water to be added to the suspension.
The above computations follow as below:
In the Levy counting chamber the dimensions of the small
squares are 1/20 mm. in width and the depth of the chamber is 1/10
mm. Therefore, the volume of each small square is 1/4,000 cubic mm.
or 1/4,000,000 cubic centimeter.
The number of spores in 5 large squares divided by 80 equals
the mean number of spores in 1/4,000,000 of 1 cc. of the diluted
suspension (1 cc. of the diluted suspension contains the spores
from 0.01 cc. of the original spore suspension).
The number of spores per cc. of the actual spore suspension
No. of spores in 5 lar gesuares
80 x 1/4,000,000 x 0.01
No. of spores in 5 large squares x 1,000, 000
20 x 0.01
5 x No. spores in 5 large squares in millions per cc.
or, since there are exactly 3 cc., the total numher of spores is 3
times the figure computed above.
Since we desire 300 miJ.lTion spores per cc., we divide the
total number of spores determined by 300 to get the volume equiva-
lent to this concentration. This will be equal to the number of
spores counted multiplied by 5/100.
The general formula for counting with the Levy counting
chamber is as follows:
Spores per cc. = No.ospores counted x 4,000,000 x dilution
The general formula for counting with chambers other than
the Lev-y counting chamner will differ, lepei.dinr, on the chamber
dimensions. For example, the Petroff-Hausser bacteria-counting
chamber uses the s..me ruling as the Levy (improved Neubauer), but
the depth of the chamber is 1/50 mm. instead of 1/10 mm. This
will change the factor in the general formula from 4,000,000 to
20,000,000, since the volume of a small square in the Petroff-
Hausser chamber is 1/20,000,000 (1/20 x 1/20 x 1/50 x 1/1,000).
The worker should acquaint himself with the chamber dimensions of
the particular make of counting chamber which he proposes to use and
determine the general formula to be used before making spore counts.
DETERMINATION OF PROPER SOIL MOISTURE TO BE USED
--IN PACKING INCUBATION BOXES
The use of soil at the proper moisture level is essential
for satisfactory survival and spore production in injected larvae.
Too low soil moistu -e will cause insufficient germination of the
seed used for food and will prevent larvae from reaching maximum
weight and hence will reduce the spore yield. Excessive soil
moisture will incrensa larval mortality by interfering with the
normal gas exchange in the soil. The absolute moisture value which
is optimum for spore production and larval survival will depend
on the soil type, sandy soils having a lower optimum value than
heavier soils. It has been found that the optimum moisture value
for a large number of soil t.,pes is that moisture which just pre-
vents adherence of soil particles to the cuticula of the larvae.
This is approximately equal to 60 percent of the "ball point" of
the soil. Determination of the "ball point" of the soil is made
as follows: T-wo 100-gram portions of air-dried soil are weighed
into containers of about 250-ml. capacity, and water is added to
them from a burette in 2-cc. increments, the soil being mixed
thoroughly after each addition until it forms a plastic mass with
just an excess of free moisture. When this point is reached, the
soil ball formed will reform when the ball is broken up and the
mass agitated by rotating the container. The amount of water added
to bring the 100-gram sample to this state is recorded as the
"ball-point" value of the soil, and 60 to 65 percent of this value
is the moisture content which the soil should contain for use in
the incubation oxess. These values are determirne.l and expressed
on the basis of grams (or cc.) of water per 100 grams of air-
FOOD REQUIREMENT FOR SMALL THIRD-STAGE LARVAE
In section 4, describing the method of preparing larv-.e for
incubation, pound of grass seed per 100 pounds of soil is speci-
fied. This amount is adequate where full-grown third instars are
used. If small or partly grown third insturs are employed, this
amount of seed is insufficient to insure maximum spore yields,
and additional amounts of seed will be required. It would be well
to use in such insta ices pound of redtop grass seed and pound
of white Dutch clover seed per 100 pounds of soil. Th, m'-re rapidly
germinating clover sead Nould promote growth faster than redtop
EQUIPMENT AND MATERIALS REQUIRED
The following equipment and materials will be required when
100,000 or more grubs are to be inoculated:
2 binocular dissecting microscopes, with paired 1OX eye-
pieces and 55mm. objectives, with microscope lamp for each.
1 compound microscope, equipped with mechanical stage, sub-
stage condenser Abbe type, 1OX eyepieces, and 16, 4, and
1.8 mm. achromatic objectives, with microscope lamp.
1 steam-pressure sterilizer for sterilizing water blanks, syr-
inges, pipettes, etc. (Sears-Roebuck 10-20 canner or simi-
lar will be suitable).
1 gas burner for sterilizer.
2 microinjectors, for injecting larvae (Dutky-Fest type). A
limited number of these can be loaned by the Bureau to
official agencies. These will have syringes, needles, and
other necessary accessories.
20 1-cc. pipettes with medium fine capillary tip.
1 0.01-cc. capillary pipette (Breed and Brew milk analysis
6 each of 1-cc., 5-cc., 10-cc. pipettes, each graduated in
2 pipette cans.
1 Levy counting chamber and coverglass, double or single rul-
ing (A. H. Thomas No 3300 or similar).
1 inoculating loop and loopholder, for filling counting cham-
ber; loop should hold approximately 0.01 cc.
50 16x150 mm. for water blanks, making up spore suspensions,
10 20x150 mm. for syringe containers.
6 wire baskets for holding test tubes.
2 battery jars.
1 oil stone, small, fine grained, for repointing needles
(Norton Pike Co. hard Arkansas oilstone, No. HB13, size 3,
1 balance or table scale, 10-30 lb. capacity.
1 platform scale, 500-lb. capacity.
1 each graduated cylinder, 100-cc., 500-cc., 1-liter.
3 volumetric flasks or dilution bottles, 200-ml.
1 Bunsen burner.
60 incubation boxes, capacity 500 grubs each, size 10-3/16" x
10-3/16" x 5-3/4" deep (inside measurement), open top
(sample can be furnished by the Bureau).
300 cross sections, capacity 100 compartments, each 1" x 1"
x 1" (sample can be furnished).
360 dividers, 10" x 10", galvanized (sample can be furnished).
1 chopper, such as meat grinder.
Mixing equipment for collecting, mixing, and drying spore-dust
mixture, similar to that shown in figure 5.
Drums, for storage of approximately 2 tons of spore-dust
Table space for injection work, not less than 4 feet of table
space for each microinjector.
Seed, redtop, 25 lb. for grub food during incubation.
Soil, approximately 2-1/2 tons, to allow 25 lb. per incubating
Carrier (for spore), talc, 2 tons; calcium carbonate, pre-
cipitated, 450 lb.
Miscellaneous trays, screens, screening racks, and sieves for
screening soil and sorting grubs. Refrigerator space for
storing infected grubs.
Figure 1.-Photograph of the Dutky-Fest microinjector with the
syringe loaded and ready for use.
Figure 2.-Photograph illustrating the method of inoculating
larvae with type A milky disease spores.
Figure 3.--Photograph of incubation boxes stacked in the
incubating chamber. Note the alternately stacked cross
sections and metal flats which divide the incubation
boxes into 500 one-inch cubes.
.: f 1
Figure 4.--Photograph of ice-packed jars of diseased
larvae stored in the refrigerator. About 100,000
larvae may be stored in the 8-cubic-foot refriger-
Figure 5.--Photograph of the experimental plant-scale equipment
used in processing diseased grubs. The letters refer to the
various elements of equipment as follows: A, 'Meat grinder
for crushing diseased larvae; B, batch mixer for incorporat-
ing crushed grub suspension with precipitated chalk; C,
hopper through which the preliminary mix is fed; D, classi-
fying chamber (note the muslin-covered blast gates for con-
trolling direction and quantity of air flow); E, cyclone
dust collector; F, high-speed paddle-wheel impeller pressure
fan; G, bag collector; H, gas-fired furnace for heating air
blast for flash drying moist dust.
UNIVERSITY OF FLORIDA
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